Research Page- Christina

Christina's Research

Christina - show some images (screenshot of BLAST result) - DR. B

Week 10:
(show image or data, not just summary or what you did) - Dr. B

• Finished Characterization
• Concentrated Protein
• FPLC
• Concentrated FPLC product

Week 9:

• Finished Expression
• Finished Purification
• Began Characterization

Week 8:

• Mini Prep Colonies
• Send to DNA sequencing
• Received Results and Blasted them
• Colony 6 worked

• Began Expression with Transformation into BL21(DE3)

Week 7:

• Received new primer and diluted it
• Restarted Cloning
• PCR off of pNIC-Bsa4 with the old gene sequence in it

• Cut accepting vector (pNIC-Bsa4)

• Cohesive End Generation
• Annealing and Transformation
• Overnight Growth

Week 6:
Christina - ok good deal. Don't bother including .docx links here - just keep those in your Google Docs. But rather show the most critical information of your results here (either data or images like the one below)

• Began Protein Purification
• Made SDS-Page Gels
• Began Protein Characterization
• Did not see our protein in the Elutions
• Decided that it was because our protein had a trans-membrane section encoded by the first few residues
• Confirmed this using TMPRED

TMPRED graph. The beginning residues are over 1000 meaning that they are probably hydrophobic.

• Ordered new Custom Forward Primers

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Week 5:

• Received results from sequencing.
• After blasting the results, both forward and reverse sequences matched the FrTuHP gene.
• Successful cloning of FrTuHP gene into pNIC-Bsa 4
• Began Protein Expression

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Week 4:

• Received results from sequencing.
• After blasting the results all the forward sequences matched the FrTuHP gene except the last 200 bp were missing.
• After also blasting the reverse sequences both colonies 2 and 3 matched all of the FrTuHP gene without any missing base pairs.

Blast results for the forward sequence.

Blast results for the reverse sequence.

• Grew up colony 2 overnight in 80mL of LB
• Spun down and Midi prep the colonies.
• Sent those results to sequencing with forward and reverse primers.
• Completed Virtual Screening Refresher

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Week 3:

• PCR amplification of FrTuHP from pUC19 (scaled up version)

Gel check of PCR. Lane 2: ladder. Land 3-7: PCR product with 4mM MgCl2. Lane 8: No DNA control.

• Cut pNIC-Bsa 4 accepting vector

Gel check for the restriction enzyme digest of the pNIC-Bsa 4 vector. Lane 2: ladder. Lane 3: uncut plasmid. Lane 4: cut pNIC-Bsa 4.

• Cohesive end generation of both accepting vector and PCR insert
• Annealing and Transformation
• Overnight growth of 4 colonies and Miniprep of those colonies
• Sent Miniprep results to sequencing with colonies 1, 2, 3, and 4 having forward primers and 2 and 3 also having reverse primers.
• Completed PyMOL refresher

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Week 2:

• Received results from sequencing but the results contained long sections of N's in the middle of the sequence.
• Restarted pNIC-Bsa 4 cloning.

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Week 1:

• Mini prep pNIC-Bsa 4 cloning colonies that were spun down over the summer.
• Sent Mini prep results to DNA sequencing

Transformation Efficiency

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Transformation of 25ng of pGBR22 plasmid with transformation efficiency of 192,000

Transformation of 5ng of pGBR22 plasmid with a transformation efficiency of 56,000

Transformation of 1ng of pGBR22 plasmid with a transformation efficiency of 152,000

RE Digest Gel

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Restriction Enzyme Digest Gel of pGFP. Lane 2: Ladder. Lane 5: EcoRI. Lane 7: PvuII. Lane 8: EcoRI + PvuII. Lane 9: uncut pGFP

PCR #1 pGBR22

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PCR gel of pGBR22. Lane 2: Ladder. Lane 3: A 0.3 ng of plasmid. Lane 4: B 3 ng of plasmid. Lane 5: C 30 ng of plasmid. Lane 6: D 0 ng no DNA control.

PCR #2 GREEN

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